EP2597461A2 - Process for directly measuring multiple biodegradabilities - Google Patents

Abstract

The method comprises preparing an organic sample, incubating the organic sample for 12-24 hours in a microplate with a fluorescent and/or colorimetric bioreagent and an inoculum of micro-organisms for degrading the sample, where the microorganisms are optionally prepared from lyophilized strains or from a culture of bacterial strains analysis of the absorbance-fluorescence emitted by a mixture over time, and calculating an industrial parameter from a calculated organic matter concentration. The method comprises preparing an organic sample, incubating the organic sample for 12-24 hours in a microplate with a fluorescent and/or colorimetric bioreagent and an inoculum of micro-organisms for degrading the sample, where the microorganisms are optionally prepared from lyophilized strains or from a culture of bacterial strains analysis of the absorbance-fluorescence emitted by a mixture over time, and calculating an industrial parameter from a calculated organic matter concentration. The analysis of absorbance-fluorescence comprises: measurement of an intensity of absorbance-fluorescence emitted following degradation of the sample by the inoculum of micro-organisms, where the obtained fluorescence intensity profile allows determination of the minimum measurement time by analysis of a coefficient of determination of a calibration curve associating the intensity of the fluorescence with a concentration of organic matter obtained by carrying out measurements of absorbance-fluorescence on samples of known increasing concentrations constituting a standard range and/or by comparison of the results obtained on samples of known concentrations by a usual standardized method, or deducing instantaneous rate of biodegradation profiles; and calculating a reference organic matter concentration using the intensity of absorbance-fluorescence measurement and using a correlation with a standard range or with a mathematical model. The step of measuring the absorbance-fluorescence intensity is carried out through an underside of the plate. The sample to be analyzed is collected at a treatment site, and the inoculum of microorganisms originates from a bacterial system present on the same site having a composition that is stable over time. The inoculum is optionally passed through 1.2 mu m mesh polyethersulfone filters. The measurement is carried out, at most every 15 minutes under aerobic conditions, by leaving the microplate open or covering the plate with a film allowing oxygen exchange without evaporation. The incubation is carried out for 12-24 hours. The measured fluorescence is converted into mg0 2.L -> 1>according to standard biological request oxygenates over 5 days by comparison with a standard range and by use of a mathematical model connecting the fluorescence intensity to the concentration. The mathematical equation is adjusted as a function of the fluorescence intensities measured on control solutions having known concentrations placed under the same conditions as the samples to be analyzed. The measurement is carried out under anaerobic conditions by covering each well of the microplate with paraffin and then closing the microplate using a cover. The plate is optionally turned over so that the fluorescence measurement takes place from above. The measurement time is chosen automatically such that the coefficient of determination of the calibration curve is closest to 1 over a total incubation period. The fluorescence emitted is converted to LCH 4.kg -> 1>raw matter according to a methane potential (BMP) standard according to a calculation comprising: converting the profiles of absorbance-fluorescence intensities or the profiles of instantaneous rates to gC-Acetate.Kg -> 1>using the mathematical equation originating from the linear regression of the calibration curve; or referring to a database constituted by the samples with known gC-Acetate.Kg -> 1>and BMP LCH 4.Kg -> 1>raw matter values in order to predict the methane potential in terms of LCH 4.kg -> 1>raw matter. The methane potential of unknown samples is directly estimated from the calibration curve in terms of LCH 4.kg -> 1>raw matter. Independent claims are included for: (1) a kit for direct measurement of biodegradability of organic samples; and (2) an analysis system.

Description

The present invention relates to a method for measuring the biodegradability of organic substrates based on the use of a fluorescent and / or colorimetric bioreactant to evaluate the microbial activity generated by the addition of organic substrates in a mixture of microorganisms. This process has been developed to allow measurements in both aerobic and anaerobic conditions.

The invention applies to the field of the analysis of the biodegradability of organic materials of various types, such as, for example, water and sludge treatment plant, agro-industrial effluents or livestock, industrial waste, agro -industrial and agricultural or compost.

Organic substrates can be used by microorganisms as substrates from which they derive energy and the ability to grow. Many industrial applications use these biochemical degradation reactions, under aerobic or anaerobic conditions, including: wastewater treatment, production of high value-added compounds, energy production, and agro-food production. The sources of organic substrates available are very varied: crops and crop residues, production and agro-food residues, household and urban waste, biomass including algae.

There are different methods for measuring the biodegradability of organic substrates, that is, to measure the extent to which microorganisms will be able to use these substrates to transform them. Each field of application uses its own method very generally based on the measurement of the product formed during the reaction, sometimes on the measurement of the degraded substrate. These methods are long, complex to set up and require heavy and expensive equipment.

Two industrial parameters are more commonly used to measure the biodegradability of organic substrates: demand biochemical (or biological) oxygen (BOD) and methane potential (BMP).

The amount of organic matter can be evaluated by measuring the industrial parameter that represents the biochemical (or biological) oxygen demand. BOD is the amount of oxygen required by aerobic microorganisms in water to oxidize organic matter, dissolved or suspended in water. The most commonly performed measurement is that of BOD ₅ . BOD ₅ corresponds to the biochemical or (biological) oxygen demand after 5 days of incubation of the sample at a temperature of 20 ° C. Its standard measurement protocol is described in standard NF EN 1899-1.

The second industrial parameter used is the methane potential, which corresponds to the volume of methane produced during the degradation of an organic substrate in the presence of anaerobic bacteria. The usual protocol is based on measuring the volume of methane produced from a known quantity of a test sample in the presence of a known quantity of anaerobic microorganisms. The test is conducted under favorable conditions (temperature, pH, presence of nutrients ...) for the degradation of the sample. The protocol is described in ISO 11734. The methane is then used in the form of heat or electricity at the site or injected into municipal networks. The residue of the methanisation, the digestate, can be valorised in the form of agricultural fertilizer in particular.

International demand WO2007 / 105040 relates to a device for rapid estimation of the biochemical oxygen demand of used drinking water. This device consists of an immobilized microbial membrane attached to an electrode, a multimeter and a portable workstation with specially developed software. Measurement of the BOD of used drinking water through this device is faster, reproducible and efficient compared to conventional dosing methods.

The patent EP0855023B1 discloses a method for determining the biological oxygen requirements of wastewater, wherein a sample wastewater is mixed with biologically neutral dilution water to a predetermined degree of dilution. The oxygen concentration that is established is then measured in a biological bath, due to the biological reaction of a predetermined biomass with the sample of wastewater diluted in the biological bath. In this method, the sample of sewage water is added and mixed in the biological bath, filled with oxygen-saturated dilution water, in a predetermined quantity in discontinuous mode of operation; and the oxygen consumption which is established per unit of time is measured and the bath is rinsed with dilution water.

But these two methods have disadvantages, especially since they only apply to liquid samples, and are quite restrictive to implement.

To determine the methane potential, the usual protocol is to inoculate a number of vials containing a small amount of medium to be analyzed with anaerobic inocula in the absence of oxygen, often in a nitrogen atmosphere. These vials are then placed in a water bath or incubator whose temperature is controlled. By manually analyzing the volume and the composition of the gas emitted by chromatography, the volume of methane produced is analyzed periodically, until the cumulative methane volume curve is stabilized. The duration of analysis is 20 to 60 days. Thus, this analytical procedure requires expensive laboratory equipment, and is very complex and demanding in terms of time and work. The composition of biogas and methane produced can be analyzed only occasionally and manually, making it difficult to set up routinely within a laboratory. There is therefore a real need for an automated test that provides quality data simply, reliably and quickly.

International demand WO 2006/079733 proposes to use fluorescence to evaluate the oxidative action of bacteria on the organic matter to be degraded. A fluorescent probe penetrates the bacteria and responds to each exchange of electrons. It is therefore a direct method of assessing bacterial activity. This method only allows to evaluate the fate of an organic matter or of organic matter associations with certain soil elements and mainly concerns the analysis of liquid samples from the soil, but does not provide a means of obtaining industrial parameters in common use, such as BOD ₅ or BMP.

The article of Dudal Y. et al. (Anal Bioanal Chem., (2006), 384, 175-179. ) reproduces the content of the international application WO 2006/079733 and discloses a microplate fluorescence assay for the measurement of catabolic activity induced by dissolved organic matter (DOM). This technique allows, from glucose calibration curves, to calculate the mineralizable amount of a DOM and to classify DOMs according to their ability to react with bacteria.

The article of Christian Guyard (Water, industry and nuisance, (01/08/10), No. 334, 51-58 ) is a review of the different techniques used for the measurement of BOD ₅ and the problems inherent in these techniques. Reference is made to the subject matter of the international application WO 2006/079733 and the correlation that would exist between the measurement of fluorescence and the measurement of BOD ₅ without however specify how it is established.

• Permettre une mesure rapide et simple à mettre en oeuvre;
• Proposer une méthode applicable à de nombreux types d'échantillons organiques;
• Améliorer la sensibilité de la méthode ;
• Etendre la gamme de concentrations mesurables par le procédé ;
• Proposer une méthode permettant l'utilisation de micro-organismes adaptés à chaque application opérationnelle ;
• Proposer une méthode permettant un travail en haut-débit ;
• Fournir des résultats sous la forme de paramètres industriels connus.

An object of the invention is to overcome the disadvantages of the state of the art, and in particular to improve the following points:

• Allow a quick and easy measurement to implement;
• To propose a method applicable to many types of organic samples;
• Improve the sensitivity of the method;
• Extend the range of measurable concentrations by the process;
• To propose a method allowing the use of microorganisms adapted to each operational application;
• To propose a method allowing a work in broadband;
• Provide results in the form of known industrial parameters.

For this purpose, the invention proposes to measure the biodegradability of organic substrates by the fluorescent and / or colorimetric detection of the microbial activity generated by the addition of organic substrates in a mixture of micro-organisms, to deduce by calibration the expected value for each field of application and to quantify the organic matter present. This measurement is carried out on a format allowing the multiplicity of measurements with an automated data processing.

This invention makes it possible to measure equivalent to conventional methods of BOD ₅ and BMP, but the method can also be used to measure other potentials in the environmental field, such as a fermentative or fertilizing potential, but also in agri-food (dairy industry) and petrochemical sectors. This method can also be used to carry out the diagnosis of a bioreactor, to measure the purification performance or to study the inhibitory or toxic effects of different products (effluents, organic substrates, etc.). It can also provide results comparable to those of respirometry.

• préparation de l'échantillon,
• incubation, pendant une durée comprise entre 1 et 48 heures, avantageusement entre 12 et 48 heures, avantageusement entre 12 et 24 heures, au sein d'une microplaque, de l'échantillon avec un bioréactif fluorescent et/ou colorimétrique et un inoculum de micro-organismes susceptibles de dégrader ledit échantillon,
• analyse de l'absorbance - fluorescence émise par le mélange au cours du temps, ladite analyse d'absorbance - fluorescence comprenant les deux étapes suivantes :
  1. a) mesure d'une intensité d'absorbance - fluorescence émise suite à la dégradation de l'échantillon par l'inoculum de micro-organismes, ledit profil d'intensité de fluorescence obtenu permettant :
     • soit de déterminer le temps minimal de mesure par analyse d'un coefficient de détermination d'une courbe de calibration, ladite courbe de calibration reliant l'intensité de la fluorescence à la concentration en matière organique étant obtenue en réalisant des mesures d'absorbance - fluorescence sur des échantillons de concentrations croissantes connues constituant une gamme-étalon, et/ou par comparaison des résultats obtenus sur des échantillons de concentrations connues par une méthode habituelle « normée »,
     • soit de déduire des profils de vitesse instantanée de biodégradation,
  2. b) utilisation de l'intensité d'absorbance - fluorescence mesurée pour calculer une concentration de matière organique de référence, grâce à une corrélation soit par une gamme-étalon, soit par un modèle mathématique ledit procédé comprenant en outre une étape de calcul d'un paramètre industriel à partir de la concentration de matière organique calculée à l'étape b).

Also, the subject of the invention is a method for directly measuring the biodegradability of organic samples, comprising the following steps:

• sample preparation,
• incubation, for a period of between 1 and 48 hours, advantageously between 12 and 48 hours, advantageously between 12 and 24 hours, in a microplate, of the sample with a fluorescent and / or colorimetric bioreactant and a micro inoculum -organisms likely to degrade said sample,
• analysis of the absorbance - fluorescence emitted by the mixture over time, said absorbance - fluorescence analysis comprising the following two steps:
  1. a) measuring an intensity of absorbance - fluorescence emitted following the degradation of the sample by the inoculum of microorganisms, said fluorescence intensity profile obtained allowing:
     • or to determine the minimum measurement time by analyzing a coefficient of determination of a calibration curve, said calibration curve connecting the intensity of the fluorescence to the organic matter concentration being obtained by performing absorbance measurements - fluorescence on samples of known increasing concentrations constituting a standard range, and / or by comparison of the results obtained on samples of known concentrations by a standard "normalized" method,
     • to deduce profiles of instantaneous rate of biodegradation,
  2. b) using the absorbance-fluorescence intensity measured to calculate a reference organic matter concentration, by correlation either by a standard range or by a mathematical model, said method further comprising a step of calculating an industrial parameter from the concentration of organic matter calculated in step b).

According to the invention, the instantaneous biodegradation speed profiles can be calculated by any technique known to those skilled in the art, in particular by derivative of the intensity profile. The speed profiles make it possible to select by simple reading the maximum instantaneous speed which is directly correlated to the substrate concentration.

According to the invention, the samples may be wastewater, drinking water, industrial effluents, wastewater, organic substrates, especially agro-industrial or livestock, industrial waste, agro-industrial and agricultural or compost and sewage sludge, algae ...

They can be used directly or after dilution.

In an advantageous embodiment of the invention, the measurement of the intensity of the absorbance-fluorescence is performed from below the plate.

In another advantageous embodiment of the invention, the sample to be analyzed is taken from a treatment site and the microorganism inoculum comes from a bacterial system of stable composition in the present time on the same site.

If necessary, the inoculum is passed through 1.2 μm mesh PES filters before use.

According to the invention, the microorganism inoculum may be derived from the sampling medium itself or be prepared from freeze-dried strains or from a culture of bacterial strains. These strains and cultures are commercially available and well known to those skilled in the art.

According to the invention, when the measurement is carried out under aerobic conditions, it is carried out either by leaving the microplate open or by covering it with a film allowing the exchange of oxygen without evaporation.

In an advantageous embodiment of the invention, the incubation is carried out for a period of between 1 hours and 24 hours, advantageously between 12 and 24 hours in an aerobic medium and at least equal to 10 hours in an anaerobic medium.

In another embodiment of the invention, the measurements are performed at least every hour and at most every 15 minutes.

In an advantageous embodiment of the invention, the measured fluorescence is converted into mgO ₂ .L ^-1 , unit of BOD ₅ (biological oxygen demand over 5 days), compared with a standard range.

In another advantageous embodiment of the invention, the measured fluorescence is converted into mgO ₂ .L ^-1, by using a mathematical model relating the fluorescence intensity to the concentration.

In another advantageous embodiment of the invention, the mathematical equation is adjusted as a function of the fluorescence intensities measured on control solutions of known concentrations placed under the same conditions as the samples to be analyzed.

In another advantageous embodiment of the invention the measurement is carried out under anaerobic conditions, in particular by covering each well of the paraffin microplate and then closing the microplate with a lid, the plate being turnable so that the fluorescence measurement is done from above.

According to the invention, in the case of a measurement under anaerobic conditions, the measurement time chosen in an automated manner can be the one at which the coefficient of determination of the calibration curve is the closest to 1 over a total incubation period.

• conversion des intensités de fluorescence en gC-Acétate.Kg^-1 à l'aide de l'équation mathématique issue de la régression linéaire de la courbe de calibration, ou
• référence à une base de données constituée BMP en LCH₄.Kg^-1 de matière brute pour prédire le potentiel méthane en LCH₄.Kg^-1 de matière brute.

In an advantageous embodiment of the invention under anaerobic conditions, the emitted fluorescence is converted into LCH ₄ .Kg ^-1 of raw material, according to the usual unit of expression of the methane potential according to a calculation comprising the following steps: :

• conversion of the fluorescence intensities to gC-Acetate.Kg ^-1 using the mathematical equation resulting from the linear regression of the calibration curve, or
• reference to a database consisting of BMP in LCH ₄ .Kg ^-1 of raw material to predict the methane potential in LCH ₄ .Kg ^-1 of raw material.

Advantageously, in accordance with the invention, the methane potential of unknown samples is directly estimated in LCH ₄ .Kg ^-1 of raw material from the calibration curve.

The method according to the invention can implement a kit comprising at least one microplate, at least one fluorescent and / or colorimetric bioreactant and standard solutions and / or control.

According to the invention, the absorbance-fluorescence analysis step is carried out by a system comprising a fluorescence reader adapted to the microplate format and calculation means arranged to implement said method.

The invention also relates to a method of quantifying organic matter present in a sample using the biodegradability measurement described above.

• Mise au point de la méthode : choix de l'inoculum et détermination du temps d'incubation optimal,
• Préparation de l'échantillon
• Mise en contact et incubation au sein d'une microplaque de l'échantillon avec un inoculum de micro-organismes susceptible de dégrader ledit échantillon et avec un bioréactif fluorescent et/ou colorimétrique,
• Analyse de l'absorbance et/ou fluorescence émise par le mélange au cours du temps.

Thus according to the invention, the measurement comprises several steps:

• Development of the method: choice of the inoculum and determination of the optimal incubation time,
• Sample preparation
• Contacting and incubation in a microplate of the sample with an inoculum of microorganisms capable of degrading said sample and with a fluorescent and / or colorimetric bioreactant,
• Analysis of the absorbance and / or fluorescence emitted by the mixture over time.

Inocula from different sources, media or environments, under different growing conditions, do not react in the same way when put in contact with the same substrate. In order to determine the relationship between the bacterial activity measured by the absorbance / fluorescence and the target industrial parameter, a characterization by the reference method (BOD or BMP) must be performed. This characterization is carried out on the solutions of the standard ranges used in the method of the invention, either synthetic solutions (example: glucose + glutamic acid mixing solution) or on real samples (example: suspension of biowaste milled at different concentrations) . The link between the concentrations of the standard solutions and the industrial parameter is thus defined and will be used for the quantification of the samples to be tested.

The minimum and / or optimal incubation time is determined according to the data processing method used.

1. a) mesure de l'intensité de l'absorbance - fluorescence par un détecteur, dans un temps minimal de mesure déterminé préalablement au cours de l'étape de caractérisation de l'inoculum,
2. b) utilisation des profils d'intensités ou des profils de vitesses instantanées pour calculer une concentration de matière organique de référence, grâce à une corrélation soit par une gamme-étalon, soit par un modèle mathématique, cette molécule de référence pouvant être le mélange glucose - acide glutamique pour la mesure de DBO₅ et l'acétate pour la mesure du BMP, ou tout autre molécule représentant un substrat modèle pour la biodégradation étudiée ;
3. c) la traduction de cette valeur de concentration de matière organique de référence en un paramètre industriel, à partir de la corrélation déterminée au cours de la caractérisation de l'inoculum.

Absorbance-fluorescence analysis is performed in three steps performed by an automated system:

1. a) measuring the intensity of the absorbance - fluorescence by a detector, in a minimum measurement time determined beforehand during the stage of characterization of the inoculum,
2. b) using intensity profiles or instantaneous velocity profiles to calculate a concentration of reference organic matter, through a correlation either by a standard range or by a mathematical model, this reference molecule may be the glucose mixture - glutamic acid for the measurement of BOD ₅ and acetate for the measurement of BMP, or any other molecule representing a model substrate for the studied biodegradation;
3. c) the translation of this reference organic matter concentration value into an industrial parameter, based on the correlation determined during the inoculum characterization.

In some cases, especially under anaerobic conditions, steps b) and c) are combined.

The data processing algorithm implemented is used to automate the decision of reading time and incubation and to interpret absorbance-fluorescence results in biodegradability units commonly used in the industrial environment: mgO ₂ .L ^-1 for the equivalent BOD ₅ conventionally used to evaluate the biodegradability of wastewater, LCH ₄ .kg ^-1 of raw material for the potential equivalent methane.

The invention therefore relates to a biodegradability measuring method, for obtaining a fast, simple and automated industrial indicative parameter, said parameter for quantifying the organic matter present in a sample.

• la FIGURE 1 représente schématiquement la mise en oeuvre du procédé selon l'invention en conditions aérobies ;
• la FIGURE 2 présente les dilutions d'échantillons « types » en fonction de la gamme utilisée pour des mesures en conditions aérobies ;
• la FIGURE 3 donne un exemple de composition du tampon de dilution à utiliser pour la préparation des échantillons.
• la FIGURE 4 représente schématiquement la mise en oeuvre du procédé selon l'invention en conditions anaérobies ;
• la FIGURE 5 donne un exemple de disposition des échantillons dans les puits d'une microplaque utilisée conformément à l'exemple 1;
• la FIGURE 6 donne un exemple de concentrations d'échantillons de gamme-étalon ;
• la FIGURE 7 est un graphe montrant l'évolution des coefficients de détermination de la courbe de calibration en fonction du temps d'incubation des échantillons-étalons, en conditions anaérobies conformément à l'exemple 1 ;
• la FIGURE 8 donne trois exemples de courbes d'étalonnage représentant les variations de l'intensité de fluorescence des échantillons de la gamme-étalon en fonction de la concentration en gC-Acétate.L^-1, pour trois temps d'incubation : 1 heure, 20 heures et 33 heures en conditions anaérobies conformément à l'exemple 1.
• La FIGURE 9 illustre la corrélation entre une mesure de DBO₅ classique et la mesure réalisée selon l'invention avec le kit Enverdi® conformément à l'exemple 2. La courbe noire correspond à la courbe pour laquelle la corrélation est égale à 1 (mesure Enverdi® - mesure DBO₅ normée), les courbes grises correspondent à la courbe pour laquelle la corrélation est égale à 1,3 (mesure Enverdi® = 1,3 mesure DBO₅ normée) et à la courbe pour laquelle la corrélation est égale à 0,7 (mesure Enverdi® = 0,7 mesure DBO₅ normée). (◊) représentent les mesures réalisées sur les échantillons conformément à l'exemple 2.

Other features and advantages of the invention will emerge from the detailed description and the attached drawings in which:

• the FIGURE 1 schematically represents the implementation of the method according to the invention under aerobic conditions;
• the FIGURE 2 presents the "typical" sample dilutions according to the range used for measurements under aerobic conditions;
• the FIGURE 3 gives an example of the composition of the dilution buffer to be used for sample preparation.
• the FIGURE 4 schematically represents the implementation of the method according to the invention under anaerobic conditions;
• the FIGURE 5 gives an example of arrangement of the samples in the wells of a microplate used according to Example 1;
• the FIGURE 6 gives an example of standard-range sample concentrations;
• the FIGURE 7 is a graph showing the evolution of the coefficients of determination of the calibration curve as a function of the incubation time of the standard samples under anaerobic conditions according to Example 1;
• the FIGURE 8 gives three examples of calibration curves representing the changes in the fluorescence intensity of the samples of the standard range as a function of the concentration of gC-Acetate.L ^-1 , for three incubation times: 1 hour, 20 hours and 33 hours under anaerobic conditions according to Example 1.
• The FIGURE 9 illustrates the correlation between a conventional measurement of BOD ₅ and the measurement carried out according to the invention with the Enverdi® kit according to Example 2. The black curve corresponds to the curve for which the correlation is equal to 1 (Enverdi® measurement - _BOD5 measurement normalized), the gray curves correspond to the curve for which the correlation is equal to 1.3 (measurement Enverdi® = 1.3 _BOD5 measurement normalized) and the curve for which the correlation is equal to 0.7 (measurement Enverdi® = 0.7 _BOD5 measurement normalized). (◊) represent the measurements made on the samples according to Example 2.

The measurement support consists of a 96-well microplate with a transparent bottom. The reading is preferably from below the microplate. The advantage of this support is that it makes it possible to work on very small volumes of samples and reagents, and to perform in parallel a large number of measurements on one or more samples.

The characteristics of the plate vary according to the type of biodegradability to be measured. For aerobic biodegradability, the microplate is optionally covered with a culture film allowing the air to pass while avoiding evaporation. For anaerobic biodegradability, each well of the microplate is paraffin-coated and then the microplate is closed with a lid. Under anaerobic conditions with suspended samples, the plate is waterproof and can be turned over for reading from above so that suspended solids fall on the paraffin and do not interfere with the measurement.

Depending on the type of measurement to be performed, aerobic or anaerobic, the protocol to be implemented is different.

Aerobic biodegradability measurement protocol (eg measurement of BOD ₅ )

The steps of this protocol are summarized in the FIGURE 1 .

Selection of the inoculum

Before carrying out the method, it is necessary to select the inoculum of microorganisms to be used to carry out the measurement.

• de la disponibilité ou non sur le site de prélèvement de l'échantillon d'un effluent chargé en bactéries, de composition assez stable dans le temps, du type eau de sortie de station d'épuration des eaux usées (STEP) ou tout autre effluent industriel chargé en bactérie ;
• de la possibilité de préparer sur place un inoculum à partir de souches lyophilisées, dépendant notamment de la disponibilité de matériel de laboratoire, et la possibilité de stockage dans les conditions requises.

For aerobic biodegradability measurements, the choice of inoculum depends on:

• the availability or not of the sample collection site of an effluent loaded with bacteria, of a rather stable composition over time, of the type of waste water from a wastewater treatment plant (WWTP) or any other effluent industrial charged with bacteria;
• the possibility of on-site preparation of an inoculum from freeze-dried strains, depending in particular on the availability of laboratory equipment, and the possibility of storage under the required conditions.

• eau de sortie de station STEP sans traitement tertiaire de désinfection ;
• eau d'entrée de STEP diluée entre 10 et 100 fois ;
• inoculum spécifique DBO₅ en pastilles ou gélules lyophilisées ;
• suspension de biofilms ;
• autre mélange de bactéries ou souches de bactéries adaptées à l'application.

The inocula that can be used are:

• STEP station outlet water without tertiary disinfection treatment;
• STEP inlet water diluted 10 to 100 times;
• specific inoculum BOD ₅ in freeze-dried pellets or capsules;
• suspension of biofilms;
• other mixture of bacteria or bacteria strains adapted to the application.

The wide choice of inocula used to implement the method according to the invention allows an adaptation of the method to multiple applications in the field or laboratory. The skilled person will choose, in light of his knowledge the adapted inoculum.

Characterization of the inoculum

• soit des solutions synthétiques contenant des concentrations croissantes d'un substrat (par exemple un mélange glucose-acide glutamique, de la cellulose, de l'acétate de sodium...)
• soit des préparations d'un échantillon à différentes dilutions
• soit des préparations de plusieurs échantillons de matrices différentes

A preliminary phase of study and characterization of the inoculum is necessary in order to determine the link between the evolution of the absorbance-fluorescence intensity and the industrial parameter that one wishes to express. This step consists of analyzing in parallel in the operating conditions of the invention and according to the reference method with the same inoculum:

• either synthetic solutions containing increasing concentrations of a substrate (for example a glucose-glutamic acid mixture, cellulose, sodium acetate, etc.)
• or preparations of a sample with different dilutions
• either preparations of several samples of different matrices

Comparison of intensity profiles or fluorescence evolution rate profiles and measurement of BOD ₅ will serve to relate the standard range to the industrial parameter, and thus to the quantification of the samples analyzed according to the method of the invention, to this inoculum.

Selection of incubation time

After the choice of one or more inocula, a second phase of adaptation of the implementation of the method according to the invention consists in determining the incubation time.

• des mesures selon le procédé de l'invention ; dans ce cas, une analyse est réalisée toutes les 30 minutes, sur une durée totale d'incubation de 24h.
• des mesures du paramètre industriel, comme par exemple une mesure DBO₅ selon la norme.

Two types of analysis are performed in parallel on real samples and synthetic control solutions in increasing concentrations corresponding to the calibration range:

• measurements according to the method of the invention; in this case, an analysis is performed every 30 minutes, over a total incubation period of 24 hours.
• measurements of the industrial parameter, such as for example a BOD ₅ measurement according to the standard.

Then, a comparison is made between the results obtained for each type of measurement. For each incubation time of the method according to the invention, the quality of the calibration curve, that is to say either the value of the coefficient of determination of the calibration curve, or the maximum instantaneous velocities measured, and the calculated concentrations of the samples and control points are recorded. The comparison of the results obtained for each incubation time with the reference method makes it possible to determine the incubation time at which both a good correlation of the standard range and a good prediction of the industrial parameter of the samples and control points are obtained. . For example, the incubation time chosen is that at which the coefficient of determination of the calibration curve is equal to 0.98 and the standard error of prediction between a potential value measured according to a standardized method and a value measured according to the process is less than 30%.

If several inocula have been tested, the one that gives the best results in the shortest incubation time is selected.

Sample preparation protocol

The samples to be analyzed undergo a simple homogenization before sampling, and dilutions according to their concentration and the ranges used.

The table presented in the FIGURE 2 presents the dilutions of the "type" samples according to the range used, for information purposes. Other ranges can be developed according to the needs of users.

The sample is diluted in distilled water or in a buffer, an example of which is given in the table of the FIGURE 3 .

Protocol for filling the microplate

• V₁ µL de réactif
• V₂ µL d'inoculum
• V₃ µL d'échantillon, de solution standard ou de solution de contrôle.

The wells of a microplate with a transparent bottom are filled according to the following protocol:

• V ₁ μL of reagent
• V ₂ μL of inoculum
• V ₃ μL of sample, standard solution or control solution.

The optimization of the volumes and concentrations of reagents makes it possible to increase the sensitivity of the method, to reduce the reading and incubation time and to improve the correspondence with conventional methods.

Reagent

The reagent is a solution of a fluorescent and / or colorimetric bioreactive indicator of microbial proliferation diluted in a buffer.

A fluorescent probe used is, for example, a fluorescent redox developer sensitive to the catabolism of the organic matter of the sample. The fluorescent bioreactant may be chosen in particular from the group consisting of commercial compositions of resazurin derivatives (sold in particular under the Alamarblue ™ products, see patent US 5,501,959 , or MTT, or PrestoBlue ™). Alamarblue ™ has the dual advantage of color change but also the formation of a fluorescent product. The color change between the non-fluorescent oxidized blue form and the fluorescent, violet reduced form is marked and is observed with the naked eye. The selected fluorescent reagent chosen for its sensitivity and for its ability to reveal all metabolisms (aerobic or anaerobic).

The dilution buffer is chosen so as not to interfere with the measurement. It can be prepared according to the protocol presented in the table of the FIGURE 3 .

The dilution rate of the fluorescent and / or colorimetric bioreactant in the buffer is defined according to the applications and the measurement ranges.

According to a particular embodiment of the process according to the invention, the pH of the solution 1 can be adjusted, in a range varying between 6.5 and 8.5.

According to another embodiment of the invention, the bioreactant is diluted only in solution 1 ( FIGURE 3 ).

The volume V ₁ of diluted reagent 1 is between 10 and 180 μl, advantageously between 50 and 150 μl and preferably equal to 90 μl.

inoculum

The inoculum, selected in the first step, may be a "real" sample, for example, an input or output water of STEP, diluted or not, with distilled water or in a buffer such as that described in the table of the FIGURE 3 . It can also be a "synthetic" inoculum, in the form of lozenges or capsules of freeze-dried bacteria.

The volume of inoculum V ₂ is between 10 and 180 μl, advantageously between 50 and 150 μl and preferably equal to 90 μl.

Standard Sample / Control Solution

The volume of the solution to be analyzed which is either a diluted or non-diluted sample, or a point of the standard range, or a control solution, is between 10 μL and 180 μL, advantageously between 50 and 150 μL and preferably equal to at 90 μL.

Incubation

The incubation is done at a temperature adapted according to the potential to be measured. For BOD ₅ measurements, the incubation is carried out at a temperature of between 29 ° C and 31 ° C, preferably equal to 30 ° C. The total incubation time is between 1 hour and 24 hours, with absorbance-fluorescence measurements at a defined frequency. The frequency of measurements is maximum every 15 minutes and at least every hour. The incubation is done directly in the reader which controls the conditions of temperature and agitation.

This protocol makes it possible to acquire absorbance - fluorescence readings according to a very regular time step, in an automated way in order to follow the biodegradation continuously.

Analysis of results according to method 1

The analysis of the results takes place in three stages:

Step 1: Elaboration of the calculation method between fluorescence intensity and concentration in mgO ₂ .L ^-1

• Cas 1 : Une mesure de gamme étalon complète est réalisée en parallèle avec les mesures des échantillons de concentrations inconnues à analyser pour obtenir une courbe de calibration. Pour chaque échantillon, l'équation de la courbe de calibration représentant l'intensité de la fluorescence en fonction de la concentration en mgO₂.L^-1 est utilisée pour calculer les concentrations des échantillons analysés. Cette courbe de calibration est obtenue en définissant au préalable une relation de proportionnalité entre une concentration en glucose-acide glutamique et une valeur de DBO₅ en mgO₂.L^-1 par analyse de solutions synthétiques de glucose-acide glutamique selon la méthode normée DBO₅.
• Cas 2 : Seuls les échantillons de concentrations inconnues sont analysés et un modèle mathématique interne est utilisé pour calculer leurs concentrations. L'équation du modèle interne reliant l'intensité de fluorescence à la concentration en mgO₂.L^-1 est déterminée par l'exploitation de résultats de nombreuses expériences préalables, au minimum au nombre de quatre. Pour chaque échantillon, l'équation du modèle interne est utilisée pour déterminer les concentrations des échantillons. Des solutions de contrôle de concentrations connues peuvent éventuellement être analysées en parallèle pour vérifier la validité du modèle.
• Cas 3 : Les échantillons de concentrations inconnues ainsi que des solutions de contrôle de concentrations connues sont analysés. Les mesures d'intensité de fluorescence sur les solutions de contrôle sont utilisées comme moyens de comparaison pour ajuster l'équation du modèle mathématique utilisé dans le cas 2 précédemment cité. La nouvelle équation obtenue est utilisée pour déterminer les concentrations des échantillons analysés.

We can distinguish three cases:

• Case 1: A full standard range measurement is performed in parallel with the measurements of the unknown concentration samples to be analyzed to obtain a calibration curve. For each sample, the equation of the calibration curve representing the intensity of fluorescence as a function of the concentration of mgO ₂ .L ^-1 is used to calculate the concentrations of the samples analyzed. This calibration curve is obtained by first defining a relationship of proportionality between a glucose-glutamic acid concentration and a BOD ₅ value in mgO ₂ .L ^-1 by analysis of synthetic glucose-glutamic acid solutions according to the standard BOD method. ₅ .
• Case 2: Only samples of unknown concentrations are analyzed and an internal mathematical model is used to calculate their concentrations. The equation of the internal model linking the fluorescence intensity to the concentration in mgO ₂ .L ^-1 is determined by exploiting the results of many previous experiments, at least four in number. For each sample, the internal model equation is used to determine sample concentrations. Known concentration control solutions can optionally be analyzed in parallel to check the validity of the model.
• Case 3: Samples of unknown concentrations as well as control solutions of known concentrations are analyzed. The fluorescence intensity measurements on the control solutions are used as comparison means to adjust the equation of the mathematical model used in the case 2 previously cited. The new equation obtained is used to determine the concentrations of the samples analyzed.

The dilution factor of the samples is taken into account in the calculation of the concentrations.

Step 2: Checking the incubation time

It is useful to check, if possible, that the conditions determined in advance are well adapted to the experiment, in particular when the selected inoculum is a "real" inoculum, resulting from a sampling. Variations in the process of the WWTP or climatic disturbances can induce changes in the quantity and quality of the bacteria present in the sample, hence incubation conditions that are different from the conditions initially chosen.

• Suivi du coefficient de détermination R² de la courbe de calibration au cours du temps : la variation du R² au cours du temps est comparée avec celle obtenue pendant les premières expériences permettant de déterminer le temps d'incubation. La valeur du R² au temps d'incubation choisi est également relevée. Sa valeur doit être supérieure à 0,98. Cette méthode est valable si une gamme-étalon est passée en plus des échantillons sur la plaque.
• Vérification de l'intensité de fluorescence des solutions de contrôle : les valeurs de l'intensité de fluorescence des solutions de contrôle sont comparées avec les valeurs attendues au temps d'incubation choisi. L'écart observé doit être inférieur à 10%. Cette méthode est valable si une ou plusieurs solutions de contrôle ont été passées.
• Vérification des concentrations des solutions de contrôle : à partir de l'équation définie (calibration ou modèle interne), les concentrations des solutions de contrôle sont calculées pour chaque temps d'incubation. Les concentrations des solutions de contrôles, calculées au temps d'incubation sont comparées avec les concentrations réelles attendues. L'écart observé doit être inférieur à 10%. Cette méthode est valable si une ou plusieurs solutions de contrôle ont été passées.

The verification of the incubation time can be done according to one of the following methods:

• Follow-up of the coefficient of determination R ² of the calibration curve over time: the variation of R ² over time is compared with that obtained during the first experiments to determine the incubation time. The value of R ² at the chosen incubation time is also recorded. Its value must be greater than 0.98. This method is valid if a standard range is passed in addition to the samples on the plate.
• Verification of the fluorescence intensity of the control solutions: the fluorescence intensity values of the control solutions are compared with the values expected at the chosen incubation time. The difference observed must be less than 10%. This method is valid if one or more control solutions have been passed.
• Checking the concentrations of the control solutions: from the defined equation (calibration or internal model), the concentrations of the control solutions are calculated for each incubation time. The concentrations of the control solutions, calculated at the incubation time, are compared with the expected actual concentrations. The difference observed must be less than 10%. This method is valid if one or more control solutions have been passed.

If these verifications lead to the conclusion that the pre-defined incubation time is not the optimal time of this experiment, another incubation time must be chosen for the reading of the concentrations. This arbitrary choice is established from the observations made on the calibration curve, in particular concerning the evolution of R ² over time, as well as on the evolution of the fluorescence intensities and / or the concentrations calculated over time of the solutions. control.

Step 3: Determining the concentrations of the samples

After reading the fluorescence intensity for each sample at the chosen incubation time, the concentration in an industrial parameter, in particular in mgO ₂ .L ^-1 for a measurement of BOD ₅ , is calculated according to the determination method. described in step 1.

Analysis of results according to method 2

The analysis of the results takes place in 2 stages:

Step 1 : Elaboration of the calculation method between fluorescence intensity and concentration in mgO ₂ / L.

• Cas 1 : Une mesure de gamme étalon complète est réalisée en parallèle avec les mesures des échantillons de concentrations inconnues à analyser pour obtenir une courbe de calibration. La courbe de calibration relie la vitesse maximale d'évolution de fluorescence au cours de l'analyse à la concentration de la solution étalon, qui peut être exprimée directement avec l'unité du paramètre industriel, selon les résultats obtenus lors de la caractérisation de l'inoculum. Pour les échantillons, la vitesse maximale d'évolution de fluorescence est déterminée après un temps d'adaptation des bactéries de l'échantillon au nouveau milieu (température, nutriments, pH...) qui peut être de 1h à 5h.
• Cas 2 : Seuls les échantillons de concentrations inconnues sont analysés et un modèle mathématique interne est utilisé pour calculer leurs concentrations. L'équation du modèle interne reliant la vitesse maximum d'évolution de fluorescence à la teneur en mgO₂/L est déterminée par l'exploitation des résultats de nombreuses expériences préalables, au minimum au nombre de quatre. Pour chaque échantillon, l'équation du modèle interne est utilisée pour déterminer les concentrations des échantillons. Des solutions de contrôle de concentrations connues peuvent éventuellement être analysées en parallèle pour vérifier la validité du modèle.
• Cas 3 : Les échantillons de concentrations inconnues ainsi que des solutions de contrôle de concentrations connues sont analysés. Les mesures d'intensité de fluorescence sur les solutions de contrôle sont utilisées comme moyens de comparaison pour ajuster l'équation du modèle mathématique utilisé dans le cas 2 précédemment cité. La nouvelle équation obtenue est utilisée pour déterminer les concentrations des échantillons analysés.

We can distinguish 3 cases:

• Case 1: A full standard range measurement is performed in parallel with the measurements of the unknown concentration samples to be analyzed to obtain a calibration curve. The calibration curve relates the maximum rate of evolution of fluorescence during the analysis to the concentration of the standard solution, which can be expressed directly with the unit of the industrial parameter, according to the results obtained during the characterization of the inoculum. For the samples, the maximum rate of evolution of fluorescence is determined after a time of adaptation of the bacteria of the sample to the new medium (temperature, nutrients, pH ...) which can be from 1h to 5h.
• Case 2: Only samples of unknown concentrations are analyzed and an internal mathematical model is used to calculate their concentrations. The equation of the internal model linking the maximum rate of fluorescence evolution to the mgO ₂ / L content is determined by the exploitation of the results of many previous experiments, at least four in number. For each sample, the internal model equation is used to determine sample concentrations. Known concentration control solutions can possibly be analyzed in parallel to check the validity of the model.
• Case 3: Samples of unknown concentrations as well as control solutions of known concentrations are analyzed. The fluorescence intensity measurements on the control solutions are used as comparison means to adjust the equation of the mathematical model used in the case 2 previously cited. The new equation obtained is used to determine the concentrations of the samples analyzed.

Step 2: Determining the concentrations of the samples

After reading the fluorescence intensity for each sample and processing the data to determine the maximum rate of evolution of the fluorescence during the analysis, the concentration in one industrial parameter, in particular in mgO ₂ / L for a measurement of BOD ₅ , is calculated according to the determination method described in step 1.

Anaerobic biodegradability measurement protocol (eg, BMP measurement)

This protocol is summarized in the FIGURE 4 .

Selection of the inoculum

• de la disponibilité ou non sur le site de prélèvement des échantillons de boues de digesteurs ou d'un système bactérien anaérobie de composition assez stable dans le temps;
• de la possibilité de préparer un inoculum modèle à partir de cultures de souches, d'un cocktail de souches, d'un digesteur de laboratoire, ou de souches lyophilisées.

For anaerobic biodegradability measurements, the choice of inoculum depends on:

• the availability or not at the sampling site of digester sludge samples or an anaerobic bacterial system with a fairly stable composition over time;
• the possibility of preparing a model inoculum from strain cultures, a cocktail of strains, a laboratory digester, or freeze-dried strains.

• boues de digesteurs
• toute biomasse provenant d'un système anaérobie
• suspensions de biofilms anaérobies et/ou biofilms anaérobies
• cultures de bactéries, ou bactéries lyophilisées
• autre mélange de bactéries ou souches de bactéries adaptées à l'application.

The inocula that can be used are:

• digester sludge
• any biomass from an anaerobic system
• suspensions of anaerobic biofilms and / or anaerobic biofilms
• bacteria cultures, or freeze-dried bacteria
• other mixture of bacteria or bacteria strains adapted to the application.

After choosing the inoculum, it is important to adjust the concentration ratio Bacteria / Fluorescent Bioreactant. This is done empirically by performing the test for different concentrations and / or volumes of inoculum and different tests of concentrations on the bioreactant. These tests are conducted on the standard points, allowing us to adjust the ratio to obtain valid absorbance - fluorescence intensities. The total volume in a well can not exceed 300μL.

According to a particular embodiment of the method according to the invention, measurements can be made in parallel on known BMP samples to verify the accuracy of the results obtained according to the invention.

Characterization of the inoculum

• soit des solutions synthétiques contenant des concentrations croissantes d'un substrat (par exemple l'acétate de sodium, la cellulose...)
• soit des préparations d'un échantillon à différentes dilutions (exemple : une suspension d'un prélèvement de biodéchets ou de boues de station d'épuration à différentes concentrations)
• soit des préparations de plusieurs échantillons de matrices différentes (ex : des biodéchets, des déchets agroalimentaires, des boues de station d'épuration, des lisiers...).

A preliminary phase of study and characterization of the inoculum is necessary in order to determine the link between the evolution of the absorbance-fluorescence intensity and the BMP industrial parameter that one wishes to express. This step consists of analyzing in parallel under the operating conditions of the invention and according to the BMP reference method, with the same inoculum:

• either synthetic solutions containing increasing concentrations of a substrate (for example sodium acetate, cellulose, etc.)
• or preparations of a sample at different dilutions (example: a suspension of a collection of biowaste or sewage sludge at different concentrations)
• or preparations of several samples of different matrices (eg bio-waste, agro-food waste, sewage sludge, manure ...).

Comparison of intensity profiles or fluorescence evolution rate profiles and industrial parameters obtained by the reference method will be used to relate the standard ranges to the industrial parameters, and thus to the quantification of the samples analyzed according to the method of analysis. invention for this inoculum.

Sample preparation protocol

The method according to the invention allows the analysis of samples in the liquid state and samples in the solid state.

The samples to be analyzed are taken in such a way that the sample is representative of the whole material deposit.

Each sample is then mixed using a food mixer.

The sample is then suspended. For example, 20 g of solid sample is suspended in distilled water until it reaches 200 g. This mass is adapted according to the ease of suspension, availability or homogeneity of the product.

If the will is to compare the results of the analyzes according to the method of the invention with BMPs, at this stage it is desirable to measure the density of the samples, if the state of the sample allows it.

The suspension obtained is again homogenized by grinding with a food mixer.

This suspension or the raw effluent can then be diluted in 5 concentrations: 1/50, 1/100, 1/200, 1/250, 1/500, dilutions usually performed.

The measurement can thus be carried out on the raw sample or the "mother" suspension, as well as on all or a selection of diluted solutions.

Protocol for filling the microplate

The FIGURE 5 gives an example of a microplate arrangement under anaerobic conditions. The standard solutions G0 to G7 correspond to different increasing concentrations of the standard range, examples of which are given in the table. FIGURE 6 . The scheme The microplate organization is entered in the absorbance - fluorescence reader program, in terms of sample identification.

• V₁ µL de réactif A
• V₂ µL de réactif B
• V₃ µL d'échantillon, de solution étalon ou de solution de contrôle
• V₄ µL d'inoculum
• 150-200 µL de paraffine

The wells of a microplate with a transparent bottom are filled according to the following protocol:

• V ₁ μL of reagent A
• V ₂ μL reagent B
• V ₃ μL of sample, standard solution or control solution
• V ₄ μL of inoculum
• 150-200 μL of paraffin

Reagent A

Reagent A is a buffer solution of which an example of composition is given in the table of the FIGURE 3 . In this example, the pH of solution 1 is adjusted to the value of 7.2.

According to a particular embodiment of the process according to the invention, the pH of the solution 1 can also be adjusted, in a range varying for example between 6.5 and 8.5, or in another pH range adapted to the application

According to another embodiment of the process according to the invention, the bioreactant is only diluted in solution 1.

Reagent B

Reagent B is a fluorescent and / or colorimetric bioreactant indicative of microbial proliferation, diluted or not in a buffer.

The dilution rate of reagent B in a buffer is between 1 and 10. For example, for questions of sensitivity, reagent B can be used at the marketing concentration with a volume of 100 μL.

According to a particular embodiment of the process according to the invention, the volume of pure product can be slightly increased, or the product can be diluted from 50 μL to 150 μL.

Diluted or non - standard raw sample / control solution

In each well, a volume of 100 μL of sample is introduced.

inoculum

After sampling, the inoculum is filtered through PES filters (polyethersulfone) mesh 1.2 microns, in order to retain the largest particles of organic materials while allowing the bacterial cells constituting the inoculum.

According to a particular embodiment of the process according to the invention, in a concentrated or stressed culture medium, a finer filtration mesh may be used. This choice may be a means of selecting bacterial communities or bacterial strains favorable to analysis or prediction.

According to another embodiment, if the inoculum is weakly loaded with organic matter, the filtration can be avoided.

Following this first filtration phase, the inoculum is introduced diluted or not in a well, respecting a total reaction volume of between 280 μL and 300 μL.

For example, an inoculum from a water purification co-product treatment digester is introduced into the well in a volume of 30 .mu.l.

Paraffin

In order for the measurement to take place under anaerobic conditions, a drop of paraffin is deposited on the reaction liquid of each well.

According to a preferred embodiment of the process according to the invention, a volume of 150 μL to 200 μL of paraffin is deposited on the surface of each well.

Incubation

Throughout the incubation period, the fluorescence intensities are collected automatically at a determined frequency, for example every hour. At each measurement time, the intensity of fluorescence of the standard points allows to draw a calibration curve representing the correlation between fluorescence intensity and acetate concentration of the standard points or fluorescence intensity and BMP of the standard points. The incubation is done directly in the reader which controls the conditions of temperature and agitation.

Results analysis Step 1: Selection of the incubation time

Under anaerobic conditions, the selection of the incubation time is performed automatically, together with the measurement of the samples, thanks to the data processing algorithm.

The parameter followed for the selection of the analysis time of the fluorescence intensities is the coefficient of determination R ² of the corresponding calibration curve, the standard solutions G0 to G7, obtained for each measurement time.

At the end of incubation, the data analysis time is chosen for the calibration curve with the best coefficient of determination. A verification of the kinetics of fluorescence intensity over time for each sample or standard is performed and allows to remove undesirable values. For example, a decrease in fluorescence intensity, a fluorescence intensity of a sample greater than the value of the fluorescence intensity of the highest point of the standard range, may reflect fluorescence intensities. associated with fermental and non-methanogenic metabolism.

Step 2: Method of calculating the methane potential

Once the analysis time has been selected, the fluorescence intensities of the unknown concentration samples are converted to gC-Acetate.Kg ^-1 using the calibration curve.

Then reference is made to a database consisting of samples having known values of gC-Acetate.Kg ^-1 and BMP LCH ₄ .Kg ^-1 of raw material. This database makes it possible to perform the prediction for samples whose methane potential is to be predicted in LCH ₄ .Kg ^-1 of raw material. Controls on known BMP samples can be performed in parallel for verification.

According to a particular embodiment of the process according to the invention, methane potential unknown samples is directly estimated from the calibration curve and expressed in CHL ₄ .Kg ^-1 of crude material. This is possible in the case where the standards alone allow a good prediction of the methane potential of complex samples, that is to say that each standard point is characterized by a methane potential.

Other features and advantages of the invention will appear on reading the detailed description of the example below.

Example 1 Prediction of methane potential under anaerobic conditions in digester sludge

In this example, the inoculum consists of sludge from an "infinitely mixed" digester of a treatment plant.

The first step is to take inoculum and samples.

The sludge from the digester is taken at mid-height from an "infinitely mixed" reactor. Input samples are also collected.

The inoculum is stored in an incubator at 35 ° C, 55 ° C or at the digester temperature.

The samples are then prepared according to the anaerobic biodegradability measurement protocol described in the invention.

• broyage
• mise en suspension
• broyage
• dilution

Solid samples that can not be taken with a propellant of 5mL undergo the following preparation steps:

• grinding
• suspended
• grinding
• dilution

• broyage
• dilution

The so-called liquid samples that can be taken with a 5 mL propipette undergo only two preparation steps:

• grinding
• dilution

The density of the samples is then measured.

The following steps are the filling of the microplate and the preparation of the inoculum.

The filling of the microplate is defined by the manipulator who arranges the standard solutions and the samples according to a personal disposition. This arrangement is then entered into the program of the fluorescence reader. An example of a provision is schematized in the FIGURE 5 , G0 to G7 representing the solutions of the standard range.

In each well of the microplate with a transparent bottom corresponding to an analysis, 50 μl of buffer are available. If 96 wells are used then the buffer is dispensed into all wells. If only a few wells are needed, then only these wells are distributed, the remaining wells can be used for future analyzes.

In each of the selected wells are introduced 100 μL of reagent B.

Then, according to the previously agreed arrangement, 100 μL of each of the standard solutions G0 to G7 provided are placed in the corresponding wells, and 100 μL of sample (crude, diluted or not) are distributed in each well corresponding to the plan of the microplate.

Using a syringe and a syringe filter, the inoculum is passed through a 1.2 μm filter. Then 30 .mu.l of this filtered inoculum are distributed in each well to complete the reaction mixture.

In order to homogenize the mixture and to slide drops previously retained on the edges of the wells, the microplate is tapped manually against the manipulation plane.

The paraffin is melted and then removed with a propellant with a capacity of 1 ml. A volume of 150 to 200 μL of paraffin is deposited on the surface of the reaction solution in each of the wells. The microplate is then closed with a lid to ensure anaerobic conditions.

The microplate thus filled is deposited in the reader for incubation and measurement. Fluorescence intensities are collected automatically every hour. At each collection, the fluorescence intensity of the standard points G0 to G7 makes it possible to draw a curve calibration model representing the linear correlation between the fluorescence intensity and the acetate concentration of the standard points. The coefficient of determination of the linear correlation makes it possible to know the adequacy between the observed data and the model. The FIGURE 7 shows the evolution of the coefficients of determination of the calibration curve as a function of the incubation time of the standard samples. Three examples of calibration curves representing the changes in fluorescence intensity of the samples of the standard range as a function of the concentration of gC-Acetate.L ^-1 , are given in FIG. FIGURE 8 for incubation times of 1 hour, 20 hours and 33 hours. It is observed that the coefficient of determination R ² of the calibration curve improves over time. For an incubation time of 33 hours, R ² is 0.99.

An incubation time of 33 hours is automatically selected to analyze the fluorescence intensities of the samples.

Then, the fluorescence intensities of the samples of unknown concentrations are converted to gC-Acetate.L ^-1 using the equation of the calibration curve ( FIGURE 8 ). Then reference is made to a database consisting of samples with known value of gC-Acetate.L ^-1 and BMP LCH ₄ .L ^-1 of raw material. This database makes it possible to perform the prediction for samples whose methane potential is to be predicted in LCH ₄ .L ^-1 of raw material.

Of course, the invention is not limited to the example just described and many adjustments can be made to this example without departing from the scope of the invention.

The method according to the invention makes it possible to obtain BOD ₅ equivalents and BMP equivalents respectively in less than 24 hours and in less than 35 hours with an excellent correlation with respect to the conventional methods for measuring these two industrial parameters.

Example 2 Prediction of BOD ₅ in samples from an urban wastewater treatment plant

Samples are samples taken throughout the process in an urban wastewater treatment plant.

The duration of the incubation in the presence of the fluorescent bioreactant is 15h at 30 ° C on a plate covered with an aerobic film;

The input water from the 25-fold diluted treatment plant is used as the type of inoculum (microorganisms)

The incubation mixture comprises 90 μl of reagent comprising the fluorescent probe, 90 μl of inoculum and 90 μl of sample to be tested.

The reference is to a glucose / glutamic acid range of 0 to 250 mg / L.

The reading is made from below in a spectrophotometer at the following wavelengths: excitation 540nm; 600nm emission.

In parallel, the BOD ₅ of the samples is measured by a conventional standard method.

The results are given in the figure 9 .

All measurements are between -30% and + 30% with respect to the line for which the correlation coefficient is equal to 1.

The measurement according to the invention therefore makes it possible to have a very good evaluation of the quantity of organic matter present very rapidly (15 hours).

Claims (12)

A method for directly measuring the biodegradability of organic samples comprising the following steps: - preparation of the sample, incubation, for a period of between 1 and 48 hours, advantageously between 12 and 24 hours, in a microplate, of the sample with a fluorescent and / or colorimetric bioreactant and an inoculum of microorganisms capable of degrading said sample, said microorganisms being optionally prepared from freeze-dried strains or from a culture of bacterial strains, analysis of the absorbance - fluorescence emitted by the mixture over time, said absorbance - fluorescence analysis comprising the following two steps: a) measuring an intensity of absorbance - fluorescence emitted following the degradation of the sample by the inoculum of microorganisms, said fluorescence intensity profile obtained allowing: either to determine the minimum measurement time by analysis of a coefficient of determination of a calibration curve, said calibration curve relating the intensity of the fluorescence to the concentration of organic matter being obtained by performing absorbance measurements. - fluorescence on samples of known increasing concentrations constituting a standard range, and / or by comparison of the results obtained on samples of known concentrations by a usual "standardized" method, - to deduce profiles of instantaneous biodegradation speed, b) using the absorbance-fluorescence intensity measured to calculate a reference organic matter concentration, by means of a correlation either by a standard range or by a mathematical model, said method further comprising a step of calculating an industrial parameter from the concentration of organic matter calculated in step b). Process according to Claim 1, characterized in that the measurement of the intensity of the absorbance-fluorescence is carried out from below the plate. Process according to any one of the preceding claims, characterized in that the sample to be analyzed is taken from a treatment site and the microorganism inoculum comes from a bacterial system of stable composition in the present time on the same. site, said inoculum possibly being passed on PES filters of mesh 1.2 microns. Process according to any one of the preceding claims, characterized in that the measurement is carried out, advantageously at least every hour and at maximum every 15 minutes, under aerobic conditions, either leaving the microplate open or covering it with a film allowing the exchange of oxygen without evaporation. Process according to any one of the preceding claims, characterized in that the measured fluorescence is converted into mgO ₂ .L ^-1 , an expression unit of BOD ₅ according to the standard in comparison with a standard range or by use of a standard mathematical model linking fluorescence intensity to concentration. Process according to Claim 5, characterized in that the mathematical equation is adjusted as a function of the fluorescence intensities measured on control solutions of known concentrations placed under the same conditions as the samples to be analyzed. Process according to any one of Claims 1 to 4, characterized in that the measurement is carried out under anaerobic conditions, in particular by covering each well with the paraffin microplate and then closing the microplate with a lid, the plate being possibly returned so that the fluorescence measurement is done from above. Process according to Claim 7, characterized in that the automatically chosen measuring time is that at which the coefficient of determination of the calibration curve is the closest to 1 over a total incubation period. Process according to one of Claims 7 to 9, characterized in that the emitted fluorescence is converted into LCH ₄ .Kg ^-1 of raw material, in particular according to the BMP (methane potential) standard, according to a calculation comprising the following steps: conversion of the absorbance-fluorescence intensity profiles or the instantaneous velocity profiles into gC-Acetate.Kg ^-1 using the mathematical equation resulting from the linear regression of the calibration curve, or reference to a database consisting of samples with a known value of gC-Acetate.Kg ^-1 and BMP in LCH ₄ .Kg ^-1 of raw material to predict the methane potential in LCH ₄ .Kg ^-1 of raw material. Process according to Claim 9, characterized in that the methane potential of unknown samples is directly estimated at LCH ₄ .Kg ^-1 of raw material from the calibration curve. Process according to any one of the preceding claims, characterized in that it uses a kit comprising at least one microplate, at least one fluorescent and / or colorimetric bioreactant and standard and / or control solutions. Process according to any one of the preceding claims, characterized in that the absorbance-fluorescence analysis step is carried out by a system comprising a fluorescence reader adapted to the microplate format and calculation means arranged to implement said process.

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